Disorders of glycosphingolipid and sphingomyelin (SM) metabolism are significant causes of seizures, blindness and mental retardation in children. Ceramide (Cer) is the core of sphingolipids and by itself promotes cell death in the brain. Cer is therefore a prime target for therapeutic efforts to reverse neurodegenerative disease. We have observed that the Cer/SM ratio is high in embryonic neurons but low in oligodendrocytes and astrocytes, and this may relate to injury susceptibility. We will determine if Cer is regulated differently in different brain cell types by culturing different populations of rat neurons and glia and exposing them to C6-NBD fluorescent analogs of Cer, sphingomyelin (SM) and glycolipids. We expect to show that neurons have low sphingomyelinase synthase (SMS) and high SMase activity, that the opposite is true in oligodendrocytes and that astrocytes are in-between. We present a series of experiments designed to explain these differences and the selective vulnerability of neurons to oxidative stress. We will confirm the findings with selective gene transfection, metabolic lipidomic analysis and in vitro assays. We will use mass- spectrometry to determine why "anti-apoptotic" enzymes such as sphingosine kinase (SK1) generate mainly dihydro-sphingosine-1-phosphate (DHS-1-P) whereas pro-apoptotic cytokines generate a mixture of DHS-1- P and S-1-P. We will use gene expression (SMS, SK1) etc to reverse the high susceptibility of neurons to oxidative stress. We will elucidate the role of oxidized phosphatidylcholine (OxPC), detected with the E06 antibody, in abnormal sphingolipid signaling leading to neurodegeneration. We will use shotgun and HPLC- Mass Spectrometric analysis to detect novel compounds (e.g.: 3-ketodihydrosphingosine) which could activate unique signaling pathways and receptors important for brain cell health. We will determine how drugs currently used (or proposed) to treat mentally retarded children, such as inhibitors of glucosyltransferase, antioxidants, simvastatin, myriocin, and cysteamine, impact on different ceramide- related metabolic pathways. We believe that our results will show which type of drug or drug combination is most likely to provide an alternative to gene therapy and whether it will primarily affect neurons or glia. [unreadable] [unreadable] [unreadable]